Corepressor for element-1-silencing transcription factor preferentially mediates gene networks underlying neural stem cell fate decisions

Abstract

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) silences neuronal genes in neural stem cells (NSCs) and nonneuronal cells through its role as a dynamic modular platform for recruitment of transcriptional and epigenetic regulatory cofactors to RE1-containing promoters. In embryonic stem cells, the REST regulatory network is highly integrated with the transcriptional circuitry governing self-renewal and pluripotency, although its exact functional role is unclear. The C-terminal cofactor for REST, CoREST, also acts as a modular scaffold, but its cell type-specific roles have not been elucidated. We used chromatin immunoprecipitation-on-chip to examine CoREST and REST binding sites in NSCs and their proximate progenitor species. In NSCs, we identified a larger number of CoREST (1,820) compared with REST (322) target genes. The majority of these CoREST targets do not contain known RE1 motifs. Notably, these CoREST target genes do play important roles in pluripotency networks, in modulating NSC identity and fate decisions and in epigenetic processes previously associated with both REST and CoREST. Moreover, we found that NSC-mediated developmental transitions were associated primarily with liberation of CoREST from promoters with transcriptional repression favored in less lineage-restricted radial glia and transcriptional activation favored in more lineage-restricted neuronal-oligodendrocyte precursors. Clonal NSC REST and CoREST gene manipulation paradigms further revealed that CoREST has largely independent and previously uncharacterized roles in promoting NSC multilineage potential and modulating early neural fate decisions.

Fig. 1.

CoREST and REST target genes in NSCs and other neural cell types. We identified 7,033 total CoREST targets and 5,042 total REST targets in neural cell species through ChIP-chip experiments. We compared profiles of CoREST and REST target genes in NSCs with those at specific developmental stages and in individual neural cell types, including neuronal subtypes (cholinergic, medium spiny, GABAergic, and glutamatergic neurons), oligodendrocyte (OL) lineage species, neural progenitors/precursors (neuronal-oligodendrocyte progenitors, radial glia, and OL precursors), and astrocytes (21, 22). (A) Percentages of the total number of CoREST or REST target genes present in all neural species that are targets within NSCs. (B and C) Comparative profiles of CoREST (B) and REST (C) target genes in NSCs and other neural cell types.

Fig. 2.

Effects of selective REST and CoREST depletion on NSC self-renewal, proliferation, and neural fate decisions. (A) REST- and CoREST-depleted secondary clones displayed reduced self-renewal as compared with control clones. (B) Selective ablation of REST resulted in the formation of smaller secondary clones, whereas selective ablation of CoREST resulted in the formation of larger secondary clones compared with control clones. (C) Clonal lineage analysis revealed that in the control condition both neuronal-restricted and bipotent neuronal-oligodendrocyte clones were generated. By contrast, REST ablation resulted in the generation of solely neuronal-restricted clones, whereas CoREST ablation resulted in the generation of a large proportion of clones with multilineage potential, a smaller proportion of bipotent neuronal-astroglial clones and a minimal complement of neuronal-restricted clones. Bars in A–C represent the mean ± SEM of three independent biological replicates. *P < 0.05; ** and ++P < 0.01; and ***P < 0.0001.

Fig. 3.

Effects of selective REST and CoREST depletion on NSC-mediated neurogenesis and gliogenesis. (A) The percentage of β-tubulin+ clones at 2 and 7 d in vitro (DIV) reveals that REST and CoREST are differentially required for neurogenesis. Analysis of O4+ clones reveals that REST and CoREST differentially regulate OL lineage elaboration and maintenance. Analysis of GFAP-immunoreactive clones reveals that REST and CoREST also have selective effects on the generation and maintenance of ASs. Bars in A and B represent the mean ± SEM of three independent biological replicates. ***, +++, and ♦♦♦P < 0.0001. (C) Immunofluorescence microscopic analysis demonstrates the lineage composition of secondary clones derived from NSCs following selective ablation of REST or CoREST compared with the control condition. Neural lineage markers were used to identify neurons (β-tubulin, FITC), oligodendrocytes (O4, DAPI), and astrocytes (GFAP, TRITC). (Scale bar, 200 μm).